Polypeptides, cells, and methods involving engineered cd16

ABSTRACT

This disclosure describes, generally, a modified form of CD 16, genetically-modified cells that express the modified CD 16, and methods that involve the genetically-modified cells. The modified form of CD 16 can exhibit increased anti-tumor and/or anti- viral activity due, at least in part, to reduced susceptibility to ADAM17-mediated shedding upon NK cell stimulation.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/971,996, filed March 28, 2014, which is incorporated hereinby reference.

SUMMARY

This disclosure describes, generally, a modified form of CD16,genetically-modified cells that express the modified CD16, and methodsthat involve the genetically-modified cells. The modified form of CD16can exhibit increased anti-tumor and/or anti-viral activity due, atleast in part, to reduced susceptibility to metalloprotease-mediatedshedding upon NK cell stimulation.

In one aspect, therefore, this disclosure describes a cell geneticallymodified to express a CD16 polypeptide that has a membrane proximalregion and an amino acid modification in the membrane proximal region.

In another aspect, this disclosure describes a cell that includes apolynucleotide that encodes a CD16 polypeptide that has membraneproximal region and an amino acid modification in the membrane proximalregion.

In either aspect, the amino acid medication reflects an addition of oneor more amino acids, a deletion of one or more amino acids, or asubstitution of one or more amino acids compared to the wild-type aminoacid sequence of the CD16 membrane proximal region. In some of theseembodiments, the substitution of one or more amino acids includes asubstitution of the serine residue at position 197 of SEQ ID NO:1.

In either aspect, the cell can be a Natural Killer (NK) cell, aneutrophil, a monocyte, or a T cell.

In either aspect, the modified CD16 polypeptide exhibits reducedsusceptibility to ADAM17-mediated shedding compared to a wild-type CD16polypeptide.

In either aspect, the modified CD16 polypeptide exhibits reducedsusceptibility to cleavage upon NK cell stimulation compared to awild-type CD1 polypeptide.

In another aspect, this disclosure describes a method that generallyinvolves administering to a patient in need of such treatment a therapythat includes (a) administering to the patient a therapeutic NKeffector, and (b) administering to the patient the any embodiment of thegenetically-modified cell summarized above.

In some embodiments, the therapeutic NK effector includes a therapeuticagent. In some of these embodiments, the therapeutic agent can includean antibody, or a therapeutic antibody fragment. In some of theseembodiments, the antibody, or antibody fragment, specifically binds to aviral antigen. In other embodiments, the antibody, or antibody fragment,specifically binds to a tumor antigen.

In some embodiments, the therapeutic agent can include a bi-specifickiller engager (BiKE) or a tri-specific killer cell engager (TriKE).

In yet another aspect, this disclosure describes a method for improvingimmunotherapy to a patient, in which the immunotherapy involvesadministering to the patient a therapeutic NK effector. Generally themethod includes further administering to the patient any embodiment ofthe genetically-modified cell summarized above.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Location of ectodomain cleavage sites in human CD16. (A) Trypticpeptides of soluble CD16 immunoprecipitated from the cell supernatant ofPMA-activated human NK cells or neutrophils were subjected to massspectrometry analysis. Four high confidence peptides with non-trypticC-termini were identified: 1 peptide from soluble CD16 released by NKcells (Peptide #1, upper left) and 3 peptides from soluble CD16 releasedby neutrophils (Peptide #2, lower left; Peptide #3, upper right; andPeptide #4, lower right). (B) Illustration of Peptides #1-4 (underlined)and putative cleavage sites (arrowheads) in CD16a (SEQ ID NO:1) andCD16b (SEQ ID NO:2). Amino acid 176 distinguishes CD16a (F) from CD16b(V) in the identified peptides. Amino acids 1-16 indicate a predictedsignal sequences of CD16a and CD16b. Amino acids 210-229 indicate thetransmembrane region of CD16a. Amino acid numbering begins withmethionine in the signal sequence. The amino acid sequences of CD16a andCD16b are from the NCBI reference sequences NM_000569.6 and NM_000570.4,respectively.

FIG. 2. Schematic illustration of CD16 ectodomain shedding, the cleavageregion, and the engineered serine-197 to proline mutation. CD16a andCD16b undergo ectodomain shedding by ADAM17 within a membrane proximalregion, as indicated. The CD16 cleavage region within the membraneproximal region is based on mass spectrometry analysis that revealedthree distinct cleavage sites in close proximity (arrowheads).Site-directed mutagenesis was performed to substitute serine-197 in CD16(amino acids 190-202 of SEQ ID NO:1) with a proline (CD16/S197P).

FIG. 3. Effects of the engineered S197P mutation on CD16a and CD16bshedding. Transfected HEK293 (human embryonic kidney) cells separatelyexpressed CD16b and CD16b/S197P (A) or CD16a and CD16a/S197P (B) atsimilar levels, as determined by flow cytometry (left panels). Thedifferent transfectants were treated with or without PMA (15 ng/ml for30 minutes at 37° C.) and soluble levels of CD16 in the mediasupernatant were quantified by ELISA (right panels). Each treatmentcondition was repeated three times for each experiment and the data arerepresentative of three independent experiments. Bar graphs showmean±SD. Statistical significance is indicated as ***P<0.001. (C)Transfected HEK293 cells expressed L-selectin (CD62L) or L-selectin andCD16b/S197P. Surface levels of L-selectin and CD16b/S197P on transfectedand mock-transfected cells were measured using flow cytometry (histogramplots). Transfectants expressing L-selectin or L-selectin andCD16b/S197P were incubated in the presence or absence of PMA for 30minutes at 37° C., and the mean fluorescence intensity (MFI) ofL-selectin staining determined (bar graph). Each treatment condition wasrepeated three times for each experiment and the data are representativeof two independent experiments. Bar graphs show mean±SD. Statisticalsignificance is indicated as *P<0.05. For all histogram plots, thex-axis=Log 10 fluorescence and the y-axis=cell number.

FIG. 4. Effects of the engineered S197P mutation on CD16a shedding in NKcells. NK92 cells transduced with empty vector (vector only), CD16a, orCD16a/S197P were treated without (Unstim.) or with PMA (100 ng/ml) for30 minutes at 37° C. (A), with IL-12 and IL-18 (100 ng/ml and 400 ng/ml,respectively) for 24 hours at 37° C. (B), or with Raji cells andrituximab for 60 minutes at 37° C. (C). Cell surface levels of CD16awere determined by flow cytometry. Isotype-matched negative controlantibody staining is indicated by a dotted line. (D) Parent NK92 cellsand transduced cells expressing CD16a or CD16a/S197P were treated withRaji cells and rituximab in the presence or absence of the ADAM17inhibitor BMS566394 (5 μM) for 60 minutes at 37° C. Soluble CD16a levelswere determined by ELISA. Each treatment condition was repeated threetimes and the data are representative of three independent experiments.Bar graphs show mean±SD. Statistical significance is indicated as***P<0.001. (E) NK92 cells expressing CD16a or CD16a/S197P were stainedwith the anti-ADAM17 mAbs M220, 623, 633, or an isotype-matched negativecontrol antibody, as indicated. (F) CD56⁺CD45⁺ NK cells derived frommock-transduced iPSCs (left panel) or iPSCs expressing recombinant CD16aor CD16a/S197P (right panels) were incubated with or without K562 targetcells for four hours at 37° C. For all histogram plots, the x-axis=Log10 fluorescence, the y-axis=cell number, and the data are representativeof at least 3 independent experiments.

FIG. 5. Effects of the engineered S197P mutation on CD16a function. (A)NK92 cells expressing CD16a or CD16a/S197P at equivalent levels (leftpanel) were treated with monomeric human IgG (0-20 μg/ml). As controls,cells were also treated with monomeric human IgA (20 μg/ml), and NK92parent cells were treated with IgG (20 μg/ml) (bar). Antibody bindingwas determined by flow cytometry, as described in Materials and Methods.The bar graph shows mean±SD of at least three separate experiments.Statistical significance is indicated as *P<0.05 versus IgG (0 μg/ml),IgA, or NK92 parent cells+IgG. (B) Mock transduced NK92 cells or NK92cells expressing CD16a or CD16a/S197P were incubated in the absence(Unstim.) or presence of Raji cells treated with or without anti-CD20rituximab for the indicated time points at 37° C. NK92 cell activationwas assessed by the up-regulation in CD107a staining by flow cytometry.For the histogram plots, the x-axis=Log 10 fluorescence and they-axis=cell number. Data are representative of at least 3 independentexperiments.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes, generally, a modified form of CD16a,genetically-modified cells that express the modified CD16a, and methodsthat involve the genetically-modified cells. The modified form of CD16acan exhibit increased anti-tumor and/or anti-viral activity due, atleast in part, to reduced susceptibility to metaaloprotease-mediatedshedding upon NK cell stimulation.

In contrast to many solid cancer types, the survival rate of women withepithelial ovarian cancer has changed little in the last 30 years.Moreover, current standard therapies for recurrent ovarian cancerprovide a low (<20%) response rate. Despite ubiquitous HER2overexpression by ovarian cancer samples, treatment with the anti-HER2antibody trastuzumab provides only limited responses in patients withadvanced ovarian cancer. This resistance to trastuzumab may arise fromdysfunctional NK cell-mediated antibody-dependent cell cytotoxicity.Thus, there is an urgent need for innovative therapeutic strategies. Wedescribe a novel approach for providing therapeutic treatment strategy.

One concern with ovarian cancer is that the milieu in which tumor cellsdevelop can be highly pro-inflammatory, and thus likely to promote CD16acleavage on infiltrating NK cells and consequently diminishingantibody-dependent cell cytotoxicity. Several antibodies have emerged aseffective targeted therapies for treating human malignancies. Theirefficacy is due in part to antibody interactions with FcγRIIIa/CD16a onNatural Killer (NK) cells and induction of cancer cell killing byantibody-dependent cell cytotoxicity. Human IgG Fc receptor CD16(FcγRIII) consists of two isoforms: CD16a (FcγRIIIa) and CD16b(FcγRIIIb). CD16a is expressed by Natural Killer (NK) cells and CD16b isexpressed by neutrophils. NK Cell activation results in a rapiddown-regulation in the surface levels of both isoforms of CD16 by aprocess referred to as ectodomain shedding—a proteolytic event thatinvolves the metalloprotease ADAM17 and occurs at a single extracellularregion proximal to the plasma membrane (FIG. 1A).

As noted above, ovarian cancer patients may be resistant to NKcell-mediated immunotherapies—i.e., the tumors are not sensitive to NKcell-mediated therapies. For example, ovarian cancer cells typicallyexpress the epidermal growth factor receptor HER2, yet its targetingwith the therapeutic antibody trastuzumab has provided only a limitedclinical response. This resistance may result, at least in part, fromectodomain shedding—i.e., NK cell activation by cytokines, target cellinteraction, and/or tumor infiltration can result in CD16a cleavage andimpaired antibody-dependent cell cytotoxicity. Thus, blocking theprocess of ectodomain shedding has clinical significance.

We have determined the cleavage sites of CD16a and CD16b using massspectrometry and cloned the cDNAs of CD16a and CD16b from human bloodleukocytes. Each cDNA was mutated in a directed manner to induce asingle amino acid change. Serine at location 197 was changed to aproline. (FIG. 1B). This mutation blocks the cleavage of CD16a andCD16b, and prevents their down-regulation upon cell activation. Theexpression of cleavage-resistant CD16a in ex vivo expanded NK cellsmaintain high surface levels of this IgG Fc receptor, which enhances NKcell stimulation, the efficacy of therapeutic antibodies, and cancercall killing.

ADAM17 has a number of cell surface substrates, but possesses noconsensus sequence for proteolysis that can be used to predict the siteof CD16a cleavage. Therefore, we used LC-MS-MS to determine theC-terminus cleavage site in soluble CD16 released from activated humanperipheral blood leukocytes. We observed three putative cleavagelocations in close proximity in the membrane proximal region of CD16(FIG. 2, arrowheads), a region that is identical between CD16a andCD16b. Although ADAM17 proteolysis does not require a consensussequence, the secondary structure of the cleavage region is important.In an attempt to block CD16a cleavage, we substituted serine-197 with aproline (CD16a^(197P)) to introduce a conformational change.

We identified the location of CD16 cleavage by immunoprecipitating CD16from the media supernatant of activated NK cells and, separately, fromthe media supernatant of neutrophils. The immunoprecipitated CD16 wastreated with PNGaseF to remove N-glycans, trypsin digested, and thegenerated peptides subjected to mass spectrometric analysis. Fourdifferent peptide patterns of high confidence were identified containingnon-tryptic C-termini (FIG. 1A).

For CD16 enriched from the media supernatant of activated NK cells, weobserved only one peptide pattern, which corresponds to amino acidsglycine-174 through alanine-195 (Peptide #1, FIG. 1A) of SEQ ID NO:l.The membrane proximal regions of CD16a and CD16b have identical aminoacid sequences except for residue 176. A phenylalanine at this locationis indicative of CD16a, which was present in Peptide #1 (FIGS. 1A andB). This peptide revealed a non-tryptic P1/P1′ cleavage position atalanine-195/valine-196 (FIG. 1B).

For CD16 enriched from the media supernatant of activated neutrophils,we detected three different peptide patterns with non-tryptic C-termini(Peptides #2-4, FIGS. 1A and 1B). Peptide #2 corresponds to amino acidsglycine-174 through alanine-195 of SEQ ID NO:2, Peptide #3 correspondsto amino acids glycine-174 through valine-196 of SEQ ID NO:2, andPeptide #4 corresponds to amino acids asparagine-180 throughthreonine-198 of SEQ ID NO:2. Peptide #2 and Peptide #3 contained avaline at position 176, indicative of CD16b, and revealed P1/P1′positions at alanine-195/valine-196 and at valine-196/serine-197 (FIG.1B). Peptide #4 possessed a P1/P1′ position atthreonine-198/isoleucine-199 (FIG. 1B). Though this peptide was derivedfrom soluble CD16 from enriched neutrophils, it does not contain anamino acid at position 176 to identify the isoform (FIG. 1B).Regardless, the high confidence peptide revealed a third cleavage sitein CD16. Taken together, these findings demonstrate the presence of acleavage region in CD16 rather than a single specific cleavage site.

We further examined the cleavage region in CD16 by using site-directedmutagenesis to determine whether CD16a and CD16b cleavage could bedisrupted in cell-based assays. ADAM17 tends to prefer an a-helicalconformation in the substrate region that interacts with its catalyticsite. Moreover, proteomic studies of ADAM17 cleavage site specificitiesrevealed a very low preference for proline residues at the P1′, P2′, orP3′ positions. We therefore substituted serine-197 in the cleavageregions of CD16a and CD16b with a proline (S197P, as indicated in FIG.2).

CD16b and CD16b/S197P were separately expressed in the human kidney cellline HEK293, which does not express endogenous CD16. The HEK293transfectants expressed CD16b or CD16b/S197P at similar levels on theirsurface (FIG. 3A). High levels of CD16b were released from thetransfected HEK293, which was increased further upon their treatmentwith PMA, as determined by ELISA (FIG. 3A). However, soluble levels ofCD16b/S197P generated by untreated or PMA-treated HEK293 cells weremarkedly lower than those of CD16b (FIG. 3A).

We also examined the effects of the S197P mutation on CD16a cleavageusing the same approach. Surface expression of CD16a requiresassociation with γ chain dimmers. We therefore used HEK293 cells stablyexpressing human y chain. Comparing HEK293 transfectants expressingequivalent surface levels of CD16a or CD16a/S197P (FIG. 3B), wedetermined the soluble levels of each receptor in the media supernatantof untreated and PMA-treated cells. Again, significantly lower levels ofsoluble CD16a/S197P were observed when compared to CD16a (FIG. 3B).

To evaluate whether the engineered S197P mutation in CD16 might disruptADAM17 activity, we also transfected HEK293 cells expressing or lackingCD16b/S197P with L-selectin, a well described ADAM17 substrate normallyexpressed by leukocytes. Both transfectants expressed equivalent levelsof L-selectin, which was similarly down-regulated following theiractivation with PMA (FIG. 3C), demonstrating that the S197P mutationaffected CD16 shedding and not ADAM17 activity.

To assess the effects of the S197P mutation on CD16a shedding in NKcells, we used the human NK cell line NK92 (Gong et al., 1994, Leukemia8:652-658). These cells lack expression of endogenous CD16a, butrecombinant CD16a can be stably expressed. We transduced NK92 cells toseparately express CD16a and CD16a/S197P. Cells expressing equivalentlevels of these receptors were activated with PMA and cell surface CD16levels were examined by flow cytometry. CD16a, but not CD16a/S197P,underwent a marked down-regulation in cell surface expression (FIG. 4A).IL-12 and IL-18 are physiological stimuli of NK cells that individuallyor in combination can induce CD16a shedding. NK92 cells treated withIL-12 and IL-18 demonstrated an appreciable down-regulation in theircell surface expression of CD16a but not CD16a/S197P (FIG. 4B). Directengagement of cell bound IgG by CD16a also can induce its shedding,which we examined here by incubating NK92 cells expressing CD16a orCD16a/S197P with the CD20-positive Burkitt's lymphoma cell line Raji inthe presence or absence of the anti-CD20 mAb rituximab. Raji cellstreated with rituximab induced the down-regulation of CD16a, but notCD16a/S197P (FIG. 4C).

BMS566394 is a highly selective ADAM17 inhibitor with a potency ordersof magnitude higher for ADAM17 than for other metalloproteases.BMS566394 blocked CD16a shedding with similar efficiency as the S197Pmutation, but had no additional blocking effect on activated NK92 cellsexpressing CD16a/S197P (FIG. 4D). These findings provide furtherevidence that ADAM17 is the primary sheddase that cleaves CD16a withinits cleavage region. It is possible, however, that ADAM17 expressionlevels were not equivalent in the NK92 cells expressing CD16a orCD16a/S197P, accounting for their dissimilar shedding. We thereforestained NK92 cells expressing CD16a or CD16a/S197P with multipleanti-ADAM17 mAbs and observed identical cell surface levels (FIG. 4E).

To establish the effect of the S197P mutation on CD16a shedding byprimary NK cells, we used human iPSCs to generate engineered NK cells.We have previously reported on deriving functional NK cells from iPSCsand their similarity to peripheral blood NK cells (Knorr et al., 2013Stem Cells Transl Med. 2:274-283; Ni et al., 2014, Stem Cells32:1021-1031). CD16a and CD16a/S197P cDNA were cloned into a SleepingBeauty transposon plasmid for gene insertion and stable expression iniPSC cells, which were subsequently differentiated into mature NK cells.NK cells derived from mock transduced iPSC cells expressed low levels ofendogenous CD16a, whereas transduced CD16a and CD16a/S197P wereexpressed at higher levels (FIG. 4F). NK cell activation occurs throughvarious receptors upon their interaction with K562 cells, includingBY55/CD160, resulting in ADAM17 activation and CD16a shedding. Westimulated the iPSC-derived NK cells with K562 cells and found thatCD16a underwent a marked down-regulation in cell surface expression,whereas the expression of CD16a/S197P remained stable (FIG. 4F).

Endogenous and recombinant CD16a have sufficient affinity to bindmonomeric IgG. To examine the effects of the S197P mutation on CD16afunction, we compared the IgG binding capacities of CD16a andCD16a/S197P. NK92 cells expressing CD16a or CD16a/S197P at equivalentlevels bound IgG in a similar dose-dependent manner (FIG. 5A). Controlsconsisted of IgA binding to NK92 cells expressing CD16a or CD16a/S197P,and IgG binding to NK92 parent cells. Both occurred at essentiallybackground levels (FIG. 5A). These findings demonstrate specific andequivalent IgG binding by CD16a and CD16a/S197P.

CD16a is a potent activating receptor in NK cells, and we examinedwhether the engineered S197P mutation affected the capacity of CD16a toinduce cell activation upon engagement of antibody-treated tumor cells.NK92 cell activation was assessed by measuring the up-regulation ofCD107a, which occurs very rapidly upon degranulation and is a sensitivemarker of NK cell activation. Mock transduced NK92 cells incubated withRaji cells treated with or without rituximab demonstrated low level andsimilar up-regulation CD107a (FIG. 5B). NK92 cells expressing CD16a orCD16a/S197P at equivalent levels when incubated with Raji cells alonemarginally up-regulated CD107a as well, whereas their incubation withRaji cells treated with rituximab resulted in a considerableup-regulation of CD107a (FIG. 5B). Taken together, the above findingsindicate that the engineered S197P mutation in CD16a did not impair itsfunction.

Thus, we show that the engineered S197P mutation in CD16a and CD16beffectively blocked their shedding in cell-based assays that involvednative ADAM17. The S197P mutation in CD16a also blocked shedding of thereceptor in the human NK cell line NK92, but it did not impair receptorfunction. NK92 cells expressing equivalent levels of CD16a orCD16a/S197P bound monomeric IgG with similar efficiency over a range ofantibody concentrations. In addition, NK92 cells expressing CD16a orCD16a/S197P up-regulated the activation marker CD107a in a comparablemanner upon their engagement of rituximab bound to Raji cells.

Pluripotent stem cells allow genetic manipulation to generate engineeredNK cells. This disclosure describes the generation of engineered NKcells from transduced iPSCs expressing wild-type CD16a or CD16a/S197P.As with NK92 cells, CD16a underwent shedding in the iPSCs-derived NKcells, demonstrating normal ADAM17 activity upon cell activation,whereas CD16a/S197P was not shed.

CD16a and NK cell cytotoxic function can undergo a considerabledown-regulation in cancer patients. The cDNAs encoding CD16a/S197P canbe used to generate stable human induced pluripotent stem cells (iPSCs)and embryonic stem cells (ESCs). These stem cells can then bedifferentiated into primary NK cells that express CD16a/S197P. Othercell populations that express cleavage resistant CD16a/S197P (e.g.,monocytes) or CD16b/S197P (e.g., neutrophils) also can be derived fromhESCs/iPSCs.

To generate an NK cell immunotherapy to be used in human patientsagainst various forms of cancer or infection, the CD16a/S197P-expressingNK cells can mediate increased antibody-dependent cell cytotoxicity(ADCC) activity or other CD16a-mediated activity (e.g., IFNγ and TNFαproduction). For example, the CD16a/S197P-expressing NK cells may becombined with therapeutic antibodies (e.g., trastuzumab or rituximab), abi-specific killer engager (BiKE, e.g., CD16×CD33, CD16×CD19, orCD16×EP-CAM bi-specific killer cell engager) or a tri-specific killercell engager (TriKE). Other therapeutic cell populations (e.g.,neutrophils, monocytes, T cells, etc.) also can be produced withincreased CD16-mediated activity.

Expression of CD16a/S197P in human iPSCs or human ESCs can produce an NKcell population with enhanced ADCC activity against neoplasticconditions such as, for example, HER2 ovarian cancer. In some cases, theneoplastic condition may be treated with a therapeutic antibody such as,for example, trastuzumab. Mature NK cells may be derived from humanembryonic stem cells and iPSCs.

Wild-type CD16a and/or CD16a/S197P can be cloned to generate a stableiPSC line or a stable ECS line expressing the individual CD16areceptors. Any suitable cloning method may be used. Exemplary cloningmethods include, for example, viral-based methods, transposon vectors(e.g., Sleeping Beauty), or nucleofection. In one example, iPSCs may bemodified using the Sleeping Beauty transposon vector. The vector cancontain a selection system such as, for example, GFP/zeocin resistancefusion protein, which allows a dual selection system (zeocin resistanceand flow cytometric sorting). The iPSCs can be differentiated intomature NK cells, as previously described (Ni et al., 2011, J. Virol.85:43-50; Knorr et al. 2013, Stem Cells Transl Med 2:274-283; Woll etal., 2009, Blood 113:6094-6101). Expression of transgenic receptors iniPSCs can lead to a high level of expression in the derived NK cells.CD16 expression in undifferentiated iPSCs may disrupt NK celldifferentiation. In such cases, CD16 expression may be delayed using,for example, a CD56 or a natural CD16a promoter, so that CD16 expressionbetter coincides with normal NK cell differentiation.

One can compare NK cells expressing equivalent levels of wild-type CD16aversus CD16a/S197P. Expression levels of the CD16 constructs can bematched by FACS sorting based on GFP expression, which occurs in aproportional manner to the CD16 constructs. Matched CD16a levels can beverified by FACS. NK cell cytotoxicity against HER2-expressing ovariancancer cells can be assessed by a standard chromium release assay in thepresence or absence of a therapeutic antibody such as, for example,trastuzumab. Antibody-dependent cell cytotoxicity with non-chromiumlabeled ovarian cancer cells can be evaluated. One can evaluate NK cellproduction of cytokines (e.g., IFNγ, TNFα) and soluble levels of CD16aby ELISA, and the cell surface levels of CD16a and other activationmarkers (e.g., CD107a, CD62L) by FACS.

The human tumor xenograft model described in Example 3 can be used toevaluate the anti-cancer activity of NK cells that express non-cleavableCD16a in vivo. Unlike human CD16, mouse CD16 does not undergo ectodomainshedding upon cell stimulation, and thus determining the effects ofCD16a shedding on NK cell-mediated ADCC cannot be modeled in normalmice. Table 1 provides a representative set of experimental groupingsand treatments.

TABLE 1 Tumor xenograft model Group n Treatment# 1 5 No treatment 2 5OVCAR3 cells only 3 5 OVCAR3 + NK cells/WT-CD16a 4 5 OVCAR3 + NK cells/WT-CD16a + trastuzumab 5 5 OVCAR3 + NK cells/CD16a^(197P) 6 5 OVCAR3 +NK cells/ CD16a^(197P) + trastuzumab 7 5 OVCAR3 + NK cells/vector only 85 OVCAR3 + NK cells/vector + trastuzumab #Treatment performed at leasttwice and data pooled.

Tumor growth and/or regression can be monitored weekly by conventionalmethods including, for example, bioluminescent imaging, ultrasound, CT,MRI, another imaging technology, and/or weighing the mice (Woll et al.,2009, Blood 113:6094-6101). Mice also can be bled (e.g., weekly) toquantify human NK cell survival. The expression and/or cell surfacelevels of various effector function markers (e.g., IFNγ, CD16a) can beevaluated using conventional techniques such as, for example, by FACS.Mice can be followed for any suitable period such as, for example, 60days. At the time of sacrifice, internal organs (e.g., spleen, liver,lungs, kidney, and/or ovaries) can be examined for evidence ofmetastasis (e.g., by bioluminescence), as previously described (Woll etal., 2009, Blood 113:6094-6101).

Our analyses allow one to define and compare the antibody-dependent cellcytotoxicity activity and in vivo potency of iPSC-derived NK cellsexpressing wild-type CD16a versus CD16a/S197P. Thus, we describe hereina modified form of CD16a, genetically-modified cells (e.g., NK cells,neutrophils, monocytes, T cells, etc.) that express the modified CD16a,and methods that involve the genetically-modified cells. For example, NKcells expressing the modified form of CD16a, CD16a/S197P, exhibitincreased anti-ovarian cancer activity due, at least in part, to reducedsusceptibility to ADAM17-mediated shedding upon NK cell stimulation.This, in turn, increases antibody-dependent cell cytotoxicity activityupon engaging antibody-tagged cancer cells such as, for example, cancercells tagged with a therapeutic antibody. Moreover, antibody recognitionby NK cells increases contact stability with tumor cells and bolsters NKcell activity through other activating receptors, such as NKG2D.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements; the terms“comprises” and variations thereof do not have a limiting meaning wherethese terms appear in the description and claims; unless otherwisespecified, “a,” “an,” “the,” and “at least one” are used interchangeablyand mean one or more than one; and the recitations of numerical rangesby endpoints include all numbers subsumed within that range (e.g., 1 to5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Mass Spectrometry

Peripheral blood collection from healthy individuals was performed inaccordance with protocols approved by the University of MinnesotaInstitutional Review Board according to protocol #9708M00134. Humanneutrophil and NK cell isolation was performed as previously described(Wang et al., 2013, Biochim Biophys Acta. 1833:680-685; Long et al.,2010, J Leukoc Biol. 87:1097-1101; Long et al., 2012, J Leukoc Biol.92:667-672). Enriched neutrophils or NK cells (1×10⁷/ml in PBS;Mediatech, Inc. Manassas, Va.) were activated with PMA (15 ng/ml or 50ng/ml, respectively; Sigma-Aldrich, St. Louis, Mo.) for 30 minutes at37° C. Cell supernatants were filtered (0.45 μm pore size) and CD16 wasimmunoprecipitated using the mAb 3G8 (BioLegend, Inc., San Diego,Calif.) and the Pierce direct immunopreciptation kit (Thermo FisherScientific, Rockford, Ill.), according to the manufacturer'sinstructions. Purified CD16 was deglycosylated by chitin bindingdomain-tagged Remove-iT PNGase F (New England BioLabs, Inc., Ipswich,Mass.), according to the manufacturer's instructions. Briefly, 10-20 μgof purified CD16 was denatured in the presence of 40 mM DTT at 55° C.for 10 minutes and then incubated with 3 μl of REMOVE-IT PNGase F (NewEngland BioLabs, inc., Ipswich, Mass.) at 37° C. for one hour. REMOVE-ITPNGase F was then removed from the reaction using chitin magnetic beads.

CD16 was subjected to SDS-PAGE and gel bands corresponding to solubleCD16 were detected by a krypton fluorescent protein stain (Thermo FisherScientific, Rockford, Ill.), verified by CD16 immunoblot analysis ofadjacent lanes in the same gel, and were then excised and subjected tostandard in-gel digestion with trypsin. Digested peptides extracted fromthe gel were dried down and reconstituted for liquid chromatography-massspectrometry analysis in 98:2:0.01, water:acetonitrile:formic acid and≦1 μg aliquots were analyzed by mass spectrometry (VELOS ORBITRAP,Thermo Fisher Scientific, Rockford, Ill.) in a data dependent scan mode,as described previously (Lin-Moshier et al., 2013, J Biol Chem.288:355-367). Database searches were performed with Protein Pilot 4.5(AB Sciex, Framingham, Mass.), which uses the Paragon scoring algorithm(Shilov et al., 2007, Mol Cell Proteomics 6:1638-1655), against the NCBIreference sequence Homo sapiens protein FASTA database to which thecontaminant database (thegpm.org/cRAP/index, 109 proteins) was appended.Search parameters were: cysteine iodoacetamide; trypsin; instrument OrbiMS (1-3 ppm) Orbi MS/MS; biological modifications ID focus, whichincludes asparagine deamidation; a thorough search effort; and FalseDiscovery Rate analysis (with reversed database).

Generation of cDNA Expression Constructs

CD16b occurs as two allelic variants termed NA1 and NA2, differing byfour amino acids in the N-terminal portion of its extracellular region.Both allelic variants of CD16b are cleaved with similar efficiency byADAM17. For this study, we examined only the NA1 variant. There are alsotwo allelic variants of CD16a that have either a valine or phenylalanineresidue at position 176. These two allelic variants of CD16a werecleaved with similar efficiency by ADAM17. For this study, we examinedonly the valine allelic variant CD16a.

CD16a and CD16b were amplified from human leukocyte cDNA, separatelycloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, Calif.) at theBamHI and EcoRI restriction enzyme sites as previously described (Wanget al., 2013, Biochim Biophys Acta. 1833:680-685; Dong et al., 2014,Arthritis Rheumatol. 66:1291-1299). The constructs were then subjectedto Quik-Change Site-directed Mutagenesis (Agilent Technologies, SantaClara, Calif.) according to the manufacturer's instructions to convertthe serine at position 197 to a proline in CD16a and CD16b. Allconstructs were sequenced to confirm the presence of the intendedmutation and the absence of any spontaneous mutations.

The CD16a cDNA was subsequently cloned into the bi-cistronic retroviralexpression vector pBMN-IRES-EGFP, provided by Dr. G. Nolan (StanfordUniversity, Stanford, Calif.), at the BamHI and EcoRI restriction enzymesites. The CD16a constructs were also cloned into a bicistronic SleepingBeauty transposon plasmid (pKT2-IRES-GFP:zeo) as previously described(Wilber et al., 2007, Stem Cells 25:2919-2927; Tian et al., 2009, StemCells 27:2675-2685). Briefly, wild-type CD16a and CD16a/S197P were PCRamplified using the primers: 5′-CCG GAA TTC CAG TGT GGC ATC ATG TGG CAGCTG CTC-3′ (sense, SEQ ID NO:XX) and 5′-CCG GAA TTC TCA TTT GTC TTG AGGGTC CTT TCT-3′ (antisense, SEQ ID NO:YY). EcoRI sites are underlined.The EcoRI-digested CD16a and CD16a/S197P PCR fragments were separatelycloned into pKT2-IRES-GFP:zeo. Correct CD16a orientation and sequencewere confirmed by PCR and sequencing analyses. We have previously clonedfull-length human L-selectin (CD62L) cDNA (Feehan et al., 1996, J BiolChem. 271:7019-7024; Matala et al., 2001, J Immunol. 167:1617-1623),which was transferred to the pcDNA3.1 vector at the restriction enzymesite Xba1. Full-length human FcRγ cDNA was cloned as previouslydescribed (Dong et al., 2014, Arthritis Rheumatol. 66:1291-1299), withthe modification that a pcDNA3.1 vector was used.

Generation of Cell Lines Expressing Recombinant L-Selectin, CD16a, andCD16b

HEK293 cells (a human embryonic kidney cell line) and NK92 cells (ahuman NK cell line) (ATCC, Manassas, Va.) were cultured according to thedepository's instructions. HEK293 cells were transiently transfectedwith pcDNA3.1 with or without CD16b, CD16b/S197P, and/or L-selectinusing Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. HEK293 cells stably expressing human FcRγwere transiently transfected with pcDNA3.1 with or without CD16a orCD16a/S197P by the same approach. NK92 cells were stably transduced withpBMN-IRES-EGFP with or without CD16a or CD16a/S197P by retrovirusgeneration and infection procedures described previously (Matala et al.,2001, J Immunol. 167:1617-1623; Walcheck et al., 2003, J Leukoc Biol.74:389-394; Wang et al., 2009, J Immunol. 182:2449-2457). Constructexpression was assessed by EGFP fluorescence and CD16 staining, asdetermined by flow cytometry. Human iPSCs (UCBiPS7, derived fromumbilical cord blood CD34 cells) were maintained on mouse embryonicfibroblasts (Knorr et al., 2013, Stem Cells Transl Med. 2:274-283; Ni etal., 2014, Stem Cells 32:1021-1031). Stable expression of CD16a orCD16a/S197P was performed using a Sleeping Beauty transposon system aspreviously described (Wilber et al., 2007, Stem Cells 25:2919-2927; Tianet al., 2009, Stem Cells 27:2675-2685). Briefly, iPSCs were nucleofectedwith pKT2-IRES-GFP:zeo in combination with transposase DNA innucleofector solution V (Lonza Inc., Gaithersburg, Md.) using programsetting B16. Nucleofected cells were immediately suspended in iPSCgrowth medium containing zeocin (50 μg/ml) and seeded onto mouseembryonic fibroblasts.

NK Cell Derivation from CD16a-hESC and CD16a-iPSC Cells

Hematopoietic differentiation of hESCs and iPSCs was performed aspreviously described (Ng et al., 2005, Blood 106: 1601-1603; Ng et al.,2008, Nat Protoc 3:768-776; Le Garff-Tavernier et al., 2010, Aging Cell9: 527-535). Briefly, 3000 single cells were seeded per well of 96-wellround bottom plates in BPEL media with stem cell factor (SCF, 40 ng/ml),vascular endothelial growth factor (VEGF, 20 ng/ml) and bone morphogenicprotein 4 (BMP4, 20 ng/ml). BPEL media contained Iscove's ModifiedDulbecco's Medium (IMDM, 86 ml, Invitrogen, Thermo Fisher Scientific,Inc., Waltham. Mass.), F12 Nutrient Mixture with Glutmax I (86 mL,Invitrogen, Thermo Fisher Scientific, Inc., Waltham. Mass.), 10%deionized Bovine Serum Albumin (BSA, 5 ml, Sigma-Aldrich, St. Louis,Mo.), 5% Polyvinyl alcohol (10 ml, Sigma-Aldrich, St. Louis, Mo.),linolenic acid (20 μl of 1 gm/ml solution, Sigma-Aldrich, St. Louis,Mo.), linoleic acid (20 μl of 1 gm/ml solution, Sigma), SYNTHECOL 500×solution (Sigma-Aldrich, St. Louis, Mo.), a-monothioglyceral (3.9 μl/100ml, Sigma-Aldrich, St. Louis, Mo.), Protein-free hybridoma mix II(Invitrogen, Thermo Fisher Scientific, Inc., Waltham. Mass.), ascorbicacid (5 mg/ml, Sigma), GLUTAMAX I (Invitrogen, Thermo Fisher Scientific,Inc., Waltham. Mass.), Insulin-transferrin-selenium 100× solution(Invitrogen, Thermo Fisher Scientific, Inc., Waltham. Mass.),Penicillin/streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.,Waltham. Mass.).

At day 11 of hematopoietic differentiation, spin embryoid bodies weredirectly transferred into 24-well plates with or without EL08-1D2stromal cells in NK media supplied with cytokines (Le Garff-Tavernier etal., 2010, Aging Cell 9:527-535). After 4-5 weeks of culture, singlecell suspensions were stained with APC-, PE-, FITC- andPerCP-cy5.5-coupled IgG or specific antibodies against human bloodsurface antigens: CD45-PE, CD56-APC, CD56-PE, CD16-PerCP-cy5.5,NKG2D-PE, NKp44-PE, NKp46-PE, CD158b-FITC, CD158e1/2-FITC (BDPharmingen, San Jose, Calif.), CD158a/h-PE and CD158i-PE (BeckmanCoulter, Inc., Pasadena, Calif.). Antibody stains were assessed by flowcytometry.

Cell Stimulation

HEK293 and NK92 cells in RPMI 1640 media (Mediatech, Inc., Manassas,Va.) were activated with 15 ng/ml and 100 ng/ml, respectively, PMA for30 minutes at 37° C. NK92 cells were activated with IL-12 (PeproTechInc, Rocky Hill, N.J.) and IL-18 (R&D Systems, Inc., Minneapolis, Minn.)at 100 ng/ml and 400 ng/ml, respectively, for the indicated time points.NK92 cell activation through CD16a was mediated by their incubation withthe CD20-positive Burkitt's lymphoma cell line Raji (ATCC, grownaccording to the depository's instructions) (1:1 ratio) treated with theanti-CD20 mAb rituximab (1 μg/ml) (Genentech, Inc., South San Francisco,Calif.), as described previously (Romee et al., 2013, Blood121:3599-3608). Excess rituximab was removed by washing the Raji cells.In some experiments, NK92 cells were pre-incubated for 30 minutes withthe selective ADAM17 inhibitor BMS566394 (5 μM) (Bristol-Myers SquibbCompany, Princeton, N.J.). NK cells derived from iPSCs were stimulatedwith the human erythroleukemic cell line K562 (ATCC, grown according tothe depository's instructions), as previously described (Romee et al.,2013, Blood 121:3599-3608). Briefly, iPSC-derived NK cells wereincubated with K562 target cells (2:1 ratio) for four hours at 37° C.

Antibody Binding Assay

Cell binding to monomeric human IgG and IgA (Sigma-Aldrich, St. Louis,Mo.) was performed as previously described with some modifications (Donget al., 2014, Arthritis Rheumatol. 66:1291-1299). NK92 parent cells ortransduced cells expressing CD16a or CD16a/S197P at 5×10⁶/ml in PBS wereincubated with IgG or IgA at the indicated concentrations in triplicatefor one hour at 4° C. The cells were extensively washed and incubatedwith APC-conjugated donkey anti-human Fc (heavy and light chain)antibody (Jackson Immunoresearch, West Grove, Pa.) according to themanufacturer's instructions. The cells were washed and then immediatelyanalyzed by flow cytometry.

Flow Cytometry and ELISA

For cell staining, nonspecific antibody binding sites were blocked andcells were stained with the indicated antibodies and examined by flowcytometry, as previously described (Wang et al., 2013, Biochim BiophysActa. 1833:680-685; Romee et al., 2013, Blood 121:3599-3608). Flowcytometric analysis was performed on FACSCanto and LS RII instruments(BD Biosciences, San Jose, Calif.). Human CD16 was detected by the mAbs3G8 (BioLegend, Inc., San Diego, Calif.) and DJ130c (Santa Cruz Biotech,Santa Cruz, Calif.). CD107a was detected by the mAb H4A3 (Biolegend,Inc., San Diego, Calif.). ADAM17 was detected by the mAbs M220 (Doedenset al., 2000, J Biol Chem. 275:14598-14607), 111633, and 111623 (R&DSystems, Inc., Minneapolis, Minn.). Human L-selectin was detected by themAb LAM1-116 (Ancell Corp., Stillwater, Minn.). Isotype-matched negativecontrol mAbs were used to evaluate levels of nonspecific staining. TheCD16 ELISA was performed by a custom cytometric bead assay, aspreviously described (Wang et al., 2013, Biochim Biophys Acta.1833:680-685).

Statistical Analysis

Statistical analysis was performed using Prism software (GraphPad, SanDiego, Calif.) using ANOVA and student's t test where appropriate. A pvalue of <0.05 was considered significant.

Example 2 Comparison of NK Cells Expressing Equivalent Levels of WTCD16a and CD16a^(197P) (CD16a/S197P)

Expression levels of the CD16 constructs are matched by FACS sortingbased on GFP expression (as done for NK92 cells described above, FIG.2), which occurs in a proportional manner to the CD16 constructs.Matched CD16a levels are verified by FACS for all assays. As a control,iPSC-derived NK cells modified with empty Sleeping Beauty transposonvector (expressing only GFP) are evaluated. iPSC-derived NK cellsexpress low levels of endogenous CD16a (data not shown). NK cellcytotoxicity against HER2-expressing ovarian cancer cells is assessed bya standard chromium release assay in the presence or absence oftrastuzumab. Antibody-dependent cell cytotoxicity with non-chromiumlabeled ovarian cancer cells is also performed. NK cell production ofcytokines (e.g., IFNγ, TNFα) and soluble levels of CD16a are evaluatedby ELISA. Cell surface levels of CD16a and other activation markers(e.g., CD107a, CD62L) are evaluated by FACS.

Example 3

Human Tumor Xenograft Model for Testing whether iPSC-Derived NK CellsExpressing CD16a^(197P) (CD16a/S197P) have Increased In VivoAnti-Ovarian Cancer Activity in the Presence of Trastuzumab.

A xenograft model using NOD/SCID/γc^(−/−) (NSG) mice and human ovariancancer cell lines stably engineered to express firefly luciferase forbioluminescent imaging (Geller et al., 2013, Cytotherapy 15:1297-1306)is used to test intraperitoneal (ip) delivery of NK cell activityagainst ovarian cancer cells. The OVCAR3 ovarian cancer cell line, whichover-expresses HER2, is used as the in vivo target (Hellstrom et al.,2001, Cancer Res 61:2420-2423). Sublethally-irradiated (225 cGY) NSGfemale mice are injected intraperitoneally with OVCAR3 (2×10⁵ cells)generated to express luciferase for bioluminescent imaging to quantifytumor growth or regression (Geller et al., 2013, Cytotherapy15:1297-1306). Tumors are allowed to grow for seven days before the miceget a single intraperitoneal injection of 20×10⁶ NK cells. Mice are thengiven IL-2 (5 μg/mouse) every other day for four weeks as previouslydescribed (Woll et al., 2009, Blood 113: 6094-6101) to promote in vivosurvival of NK cells. Trastuzumab is administered at a dose of 50 μgintraperitoneally once weekly for four weeks, a previously used dose inthis model (Warburton et al., 2004, Clinical cancer research10:2512-2524). The in vivo potency of iPSC-derived NK cells expressingequivalent levels of WT CD16 or CD16a^(197P) (CDa6a/S197P) are compared.Controls include iPSC-derived NK cells expressing GFP alone (vectoronly), and a cohort of mice receiving ovarian cancer cells only. Allmice get the same IL-2 treatment.

Tumor growth/regression are monitored weekly by bioluminescent imagingand weighing the mice, as previously described (Woll et al., 2009, Blood113: 6094-6101). Mice are also bled weekly to quantify human NK cellsurvival. The expression/cell surface levels of various effectorfunction markers (e.g., IFNγ, CD16a) are evaluated by FACS. Mice arefollowed for ˜60 days. At the time of sacrifice, internal organs(spleen, liver, lungs, kidney, and ovaries) are examined bybioluminescence for evidence of metastasis, as previously described(Woll et al., 2009, Blood 113: 6094-6101).

Exemplary Embodiments

Embodiment 1. A cell genetically modified to express a CD16 polypeptidethat comprises a membrane proximal region and an amino acid modificationin the membrane proximal region.

Embodiment 2. A cell comprising:

a polynucleotide that encodes a CD16 polypeptide that comprises amembrane proximal region and an amino acid modification in the membraneproximal region.

Embodiment 3. The cell of Embodiment 1 or Embodiment 2 wherein the aminoacid medication reflects an addition of one or more amino acids, adeletion of one or more amino acids, or a substitution of one or moreamino acids compared to the wild-type amino acid sequence of the CD16membrane proximal region.

Embodiment 4. The cell of Embodiment 3 wherein the substitution of oneor more amino acids comprises a substitution of the serine residue atposition 197 of SEQ ID NO:l.

Embodiment 5. The cell of any preceding Embodiment wherein the cell is aNatural Killer (NK) cell.

Embodiment 6. The cell of any preceding Embodiment wherein the cell is aneutrophil.

Embodiment 7. The cell of any preceding Embodiment wherein the cell is amonocyte.

Embodiment 8. The cell of any preceding Embodiment wherein the modifiedCD16 polypeptide exhibits reduced susceptibility to ADAM17-mediatedshedding compared to a wild-type CD16 polypeptide.

Embodiment 9. The cell of any preceding Embodiment wherein the modifiedCD16 polypeptide exhibits reduced susceptibility to cleavage upon NKcell stimulation compared to a wild-type CD16 polypeptide.

Embodiment 10. A method comprising administering to a patient in need ofsuch treatment a therapy that comprises:

-   -   administering to the patient a therapeutic NK effector; and    -   administering to the patient the cell of any one of claims 1-9.

Embodiment 11. The method of Embodiment 10 wherein the therapeutic NKeffector comprises a therapeutic agent.

Embodiment 12. The method of Embodiment 11 wherein the therapeutic agentspecifically recognizes a tumor antigen.

Embodiment 13. The method of Embodiment 12 wherein the therapeutic agentcomprises an antibody or an antibody fragment that specificallyrecognizes the tumor antigen.

Embodiment 14. The method of Embodiment 13 wherein the tumor antigencomprises HER2.

Embodiment 15. The method of Embodiment 13 or Embodiment 14 wherein theantibody comprises trastuzumab or rituximab.

Embodiment 16. The method of Embodiment 10 wherein the therapeutic NKeffector comprises a bi-specific killer engager (BiKE)

Embodiment 17. The method of Embodiment 16 wherein the BiKE comprises aCD16×CD33 BiKE, a CD16×CD19 BiKE, or a CD16×EP-CAM BiKE.

Embodiment 18. The method of Embodiment 10 wherein the therapeutic NKeffector comprises a tri-specific killer cell engager (TriKE).

Embodiment 19. The method of any one of Embodiments 11 or 16-18 whereinthe therapeutic agent specifically recognizes a viral target.

Embodiment 20. A method for improving therapy to a patient that includesadministering to the patient a therapeutic NK effector, the methodcomprising:

administering to the patient the cell of any one of claims 1-9.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A cell genetically modified to express a CD16 polypeptide thatcomprises a membrane proximal region and an amino acid modification inthe membrane proximal region.
 2. A cell comprising: a polynucleotidethat encodes a CD16 polypeptide that comprises a membrane proximalregion and an amino acid modification in the membrane proximal region.3. The cell of claim 1 wherein the amino acid modification reflects anaddition of one or more amino acids, a deletion of one or more aminoacids, or a substitution of one or more amino acids compared to thewild-type amino acid sequence of the CD16 membrane proximal region. 4.The cell of claim 3 wherein the substitution of one or more amino acidscomprises a substitution of the serine residue at position 197 of SEQ IDNO:1.
 5. The cell of claim 1 wherein the cell is a Natural Killer (NK)cell.
 6. The cell of claim 1 wherein the cell is a neutrophil.
 7. Thecell of claim 1 wherein the cell is a monocyte.
 8. The cell of claim 1wherein the modified CD16 polypeptide exhibits reduced susceptibility toADAM17-mediated shedding compared to a wild-type CD16 polypeptide. 9.The cell of claim 1 wherein the modified CD16 polypeptide exhibitsreduced susceptibility to cleavage upon NK cell stimulation compared toa wild-type CD16 polypeptide.
 10. A method comprising: administering toa patient in need of such treatment a therapy that comprises:administering to the patient a therapeutic NK effector; andadministering to the patient the cell of claim
 1. 11. The method ofclaim 10 wherein the therapeutic NK effector comprises a therapeuticagent.
 12. The method of claim 11 wherein the therapeutic agentspecifically recognizes a tumor antigen.
 13. The method of claim 12wherein the therapeutic agent comprises an antibody or an antibodyfragment that specifically recognizes the tumor antigen.
 14. The methodof claim 13 wherein the tumor antigen comprises HER2.
 15. The method ofclaim 13 wherein the antibody comprises trastuzumab or rituximab. 16.The method of claim 10 wherein the therapeutic NK effector comprises abi-specific killer engager (BiKE)
 17. The method of claim 16 wherein theBiKE comprises a CD16×CD33 BiKE, a CD16×CD19 BiKE, or a CD16×EP-CAMBiKE.
 18. The method of claim 10 wherein the therapeutic NK effectorcomprises a tri-specific killer cell engager (TriKE).
 19. The method ofclaim 11 wherein the therapeutic agent specifically recognizes a viraltarget.
 20. A method for improving therapy to a patient that includesadministering to the patient a therapeutic NK effector, the methodcomprising: administering to the patient the cell of claim 1.